Process Study And Functional Evaluation Of Compound Enzyme For Preparation Of Walnut Peptide

Walnut(walnut),rich in fat,protein,vitamins and minerals,is one of the most common nuts in the world,and walnut meal is a major by-product in the processing and production of walnut oil,and its main component is walnut protein.At present,walnut meal in China is often used as feed or directly discarded,which makes walnut meal not used efficiently.China’s walnut meal by-products are rich in sources,and the comprehensive development and utilization of walnut meal by-products has positive social and practical significance,and also has a broad market prospect.In order to make efficient use of walnut meal resources,this thesis carried out a study on the preparation of walnut peptides by composite enzymes and the evaluation of product functionality.This study carried out the screening of the most suitable protease-inducing strains;the optimization of mixed bacteria-inducing protease process and process of preparing walnut peptide by enzymatic hydrolysis of walnut meal;the research on the functional indicators of walnut peptides for antioxidant and fatigue resistance;the study on the ACE inhibitory effect of walnut peptides and the preliminary separation and purification of walnut antihypertensive peptides.The main findings are listed as follows:(1)Screening of strains producing complex proteasesBy analyzing the protease activity of four strains commonly used in production(Aspergillus niger 3.350,Aspergillus niger 3.324,Aspergillus oryzae 3.042 and Aspergillus oryzae 7801)induced by different protein substrates,it was found that walnut meal was the substrate-induced protease with the highest activity.The enzyme liquid has high acidity,neutrality and alkaline protease activity.A study on enzyme production from mixed bacteria fermentation found that the combination of Aspergillus niger 3.350 and Aspergillus niger 3.324 induced a higher overall protease activity level than the protease induced by the single bacteria,acidic protease activity increased by 37.22%,neutral protease activity increased by 72.88%,alkaline protease activity increased by 89.02%.(2)Research on optimization of protease production conditions for mixed bacteriaThrough the response surface optimization strategy,the process conditions of protease production by mixed bacteria were studied in order to further improve the enzyme activity and provide efficient enzyme preparation for the enzymatic hydrolysis of walnut meal.According to the study,the optimal conditions for enzyme production of mixed bacteria were:pH 4.9,0.017%inoculation volume and induction time 52h.Under this condition,the mixed protease induced protease activity was 4.3545U/mL,which was about 37.98%higher than that before the optimization.(3)Research on the optimization of walnut peptide enzymatic hydrolysis processIn order to improve the preparation rate of walnut peptide-hydrolysis,the conditions of walnut peptidase hydrolysis were studied by response surface optimization.During the research,the DPPH clearance rate of the proteolytic products was used as an indicator,and the optimal enzymolysis conditions of walnut meal were:pH6.7,enzyme amount 5U and induction time 3.5h.Under these conditions,the DPPH clearance rate of proteolysis products was 83.24%,which was about 9.5%higher than that before optimization.(4)Functional study of walnut peptidesAn experimental study on the anti-fatigue effect,antioxidant performance and blood pressure lowering performance of walnut peptide products were carried to explore the biological functions of walnut peptide products.It was found that the weight-bearing swimming test results showed that the swimming exhaustion time of mice in each group was significantly prolonged,the contents of blood lactic acid and serum urea nitrogen in mice were also significantly decreased with the test of walnut peptide samples,and the liver glycogen and muscle glycogen reserves were also increased,indicating that walnut peptide products had the effect of relieving fatigue.At the same time,the measured peptide content was 2.63 mg/mL and the total reducing power was 1.191.The DPPH·and ABTS·+radical scavenging rates were 83.24%and 75.32%.Respectively,the scavenging rates of reactive oxygen superoxide anion and hydroxyl radical were 62.64%69.61%.In addition,walnut peptide also showed a good strong blood pressure lowering function,and the ACE inhibition rate was measured to be 93.87%.(5)Preliminary purification of walnut antihypertensive peptidesIn this study,the antihypertensive peptide of walnut was separated and purified by dialysis and gel chromatography.First,separate the enzymolysis solution with 8 kDa dialysis bags to obtain the component A greater than or equal to 8 kDa and the component B less than 8 kDa.The ACE inhibition rates of the two components that were initially separated were determined to be 15.13%and 85.91%.The B component collected by dialysis was separated and purified again by gel chromatography.The highest ACE inhibition rate of peak 2 was 88.30%,followed by peak 3 was 74.30%,and peak 1 had the lowest ACE inhibition rate of 54.72%In this study,it was the first time to prepare walnut peptide by the way of mixed bacteria inducing enzyme production,which not only improves the economic benefits of walnut meal,but also greatly improves the yield of walnut peptide,and provides a certain technical guidance for the efficient utilization of walnut meal resources At the same time,this study also carried out a comprehensive analysis of the functionality of walnut peptides,and clarified the multiple active functions of walnut peptides,which provided theoretical support for the promotion of walnut peptide products.